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Keywords:

  • ascidian;
  • GFP;
  • CFP;
  • BFP;
  • YFP;
  • DsRed;
  • lacZ;
  • electroporation;
  • transgenic embryo;
  • gene expression

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The green fluorescent protein (GFP) is used extensively to monitor gene expression and protein localization in living cells, particularly in developing embryos from a variety of species. Several GFP mutations have been characterized that improve protein expression and alter the emission spectra to produce proteins that emit green, blue, cyan, and yellow wavelengths. DsRed and its variants encode proteins that emit in the orange to red wavelengths. Many of these commercially available fluorescent proteins have been “codon optimized” for maximal levels of expression in mammalian cells. We have generated several fluorescent protein color variants that have been codon optimized for maximal expression in the ascidian Ciona intestinalis. By analyzing quantitative time-lapse recordings of transgenic embryos, we demonstrate that, in general, our Ciona optimized variants are detected and expressed at higher levels than commercially available fluorescent proteins. We show that three of these proteins, expressed simultaneously in different spatial domains within the same transgenic embryo are easily detectable using optimized fluorescent filter sets for epifluorescent microscopy. Coupled with recently developed quantitative imaging techniques, our GFP variants should provide useful reagents for monitoring the simultaneous expression of multiple genes in transgenic ascidian embryos. Developmental Dynamics 235:456–467, 2006. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The identification of the gene encoding green fluorescent protein (GFP) from the jellyfish Aequoria victoria was first described by Prasher and colleagues (1992). GFP expression has been used extensively to study developmental processes in zebrafish, Xenopus, chickens, sea urchins, Drosophila, mice, Caenorhabditis elegans, and ascidians (reviewed by Yu et al.,2003). Several laboratories have identified point mutations that alter the spectral properties of the protein, and additional mutations have been described that alter temperature sensitivity to improve protein folding and chromophore maturation (Tsien,1998). More recently, fluorescent proteins from other cnidarians, particularly corals and sea anemones, have been discovered that emit light in the orange to red range (reviewed by Matz et al.,2002; Shaner et al.,2004). Many fluorescent protein color variants, particularly those available commercially, have been optimized for maximal protein translation in mammalian cells by inserting nearly 200 silent mutations to reduce the use of rare codons, thus eliminating constraints on protein production (Yang et al.,1996). Whereas these proteins are translated efficiently in mammalian cells, the altered codon usage may not be ideal for maximal protein expression in nonmammalian cells.

To address whether mammalian codon optimization adversely effects translation of fluorescent proteins in invertebrate embryos, we quantitatively analyzed the expression of several fluorescent proteins (CFP [cyan], GFP, and RFP [red]) in transgenic ascidian embryos. Ascidians are invertebrate chordates that are particularly advantageous for studying developmental gene regulation (reviewed by Satoh,2003; Satoh et al.,2003). In less than a single day, the embryo develops to form a ∼2,500 cell tadpole larva that has well-defined cell lineages, is relatively transparent, and the entire period of development may be recorded by high-resolution time-lapse microscopy with single cell resolution (Satoh,1994). The genomes of two closely related species have been completely sequenced (Dehal et al.,2002; and http://www-genome.wi.mit.edu/annotation/ciona), there is an extensive collection of over 650,000 expressed sequence tag (EST) sequences, and the expression patterns of several thousand genes have been examined by in situ hybridization (Nishikata et al.,2001; Satou et al.,2001;2002a,b; Fujiwara et al.,2002; Kusakabe et al.,2002). One of the strengths of using the ascidian embryo to study developmental gene regulation is the ability to create hundreds or thousands of transgenic embryos in a single day using a simple electroporation procedure (Corbo et al.,1997; Di Gregorio and Levine,2002; Zeller,2004). Importantly, ascidians and vertebrates share many of the same regulatory genes and developmental strategies required to construct a basic chordate body plan, although there is reduced genetic redundancy in the ascidian. These features make ascidian embryos an excellent system in which to study chordate gene regulation and the evolution of gene regulatory networks used during chordate development.

We report on the generation of three different fluorescent proteins (CFP, GFP, and mRFP) that have been optimized for expression in Ciona intestinalis embryos. We demonstrate that we can easily detect the expression of three different fluorescent proteins in single embryos derived from eggs that were co-electroporated with three different transgenes using defined excitation and emission filter sets for conventional epifluorescence microscopes. Several of the transgenes we created for these studies were designed using computational methods that detect putative cis-regulatory domains in ascidians (Johnson et al.,2004; Kusakabe et al.,2004; Christiaen et al.,2005). The CFP and GFP proteins were generated by introducing previously characterized point mutations (reviewed by Tsien,1998) into the wild-type Aequoria victoria GFP cDNA sequence. Several DsRed (Matz et al.,1999) variants—wild-type, human codon-optimized, and a monomeric form of DsRed called mRFP (Campbell et al.,2002)—were also examined in this study. Codon optimized variants of CFP, GFP, and mRFP, designed for efficient expression in Ciona intestinalis, were generated and the expression of these proteins was compared with the expression of nonoptimal proteins using time-lapse microscopy. In general, the Ciona optimized proteins were more readily detected and embryos expressing these proteins had higher levels of fluorescence compared with nonoptimized proteins at the end of time lapse recordings. Our fluorescent protein reagents should prove useful for studying gene expression in ascidians and the ability to detect multiple fluorescent proteins within the same living embryo provides a means for measuring the simultaneous expression of multiple genes within living cells and embryos.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Generation of Fluorescent Protein Variants in Wild-Type Aequoria Sequence Backgrounds and Expression in Transgenic Ascidian Embryos

Many of the commercially available GFP protein variants have been codon optimized for improved expression in mammalian cells because the wild-type sequence is not efficiently translated (Yang et al.,1996). We compared the codons present in several humanized fluorescent proteins (HeGFP, HDsRed) with the sequences for wild-type GFP and wild-type DsRed (all abbreviations are listed in Table 1) with the predicted codon usage for the ascidian Ciona intestinalis, generated using the program CodonW (Peden,1999) based on all of the predicted genes present in Release 1 of the C. intestinalis genome (Dehal et al.,2002). A summary of our findings, presented in Table 2, suggested that the humanized fluorescent proteins have several codons that are infrequently used in C. intestinalis, particularly for the amino acids Arg, Pro, Leu, Gly, Ala, and Ser, and, therefore, may interfere with optimal protein expression and fluorescent detection in these animals. This hypothesis was supported by comparing the expression of the first GFP variant we created, using the Aequoria sequence as a scaffold, with HeGFP, a commercially available human codon optimized GFP. Both of these GFP variants have the point mutations F64L and S65T, and we observed earlier and brighter expression of our GFP compared with HeGFP (data not shown). Because of this observation, we choose to generate all of our fluorescent protein mutants using the wild-type Aequoria sequence as a cloning scaffold.

Table 1. Fluorescent Proteins Used in This Study
VariantsDescriptionAmino acid changes (relative to GFP)
  • a

    Extraneous mutations introduced by polymerase chain reaction. GFP, green fluorescent protein.

GFP  
HeGFPHumanized eGFPF64L, S65T
aGFPM5GFP, Aequoria scaffoldF64L, S65T, V163A, I167T, S175G
CoGFPM5GFP, Ciona codon optimizedF64L, S65T, V163A, I167T, S175G
aBFPBFP, Aequoria scaffoldT9Na, F64L, Y66H, Y145F, V163A, S175G, V219Ga
aCFPCFP, Aequoria scaffoldF64L, Y66W, N146I, M153T, V163A, S175G, N212K
CoCFPCFP, Ciona codon optimizedF64L, S65T, Y66W, S72A, Y145A N146I, H148D, M153T, V163A S175G
DsRed  
 HDsRed2Humanized DsRed2 
 wtDsRedwild-type DsRed 
 HmRFPHumanized mRFP 
 ComRFPmRFP, Ciona codon optimized 
Table 2. Rare Codon Usage in Ciona intestinalis
Amino acidCodon% use in Cionaa% use in aGFPM5% use in HeGFP% use in wtDsRed% use in HDsRed2Most frequently used codon in Ciona% use in Cionaa
  • a

    Percentage of codon usage from an analysis of all predicted coding regions from Release V1 of the C. intestinalis genome (Dehal et al.,2002) using the CodonW package (Peden,1999).

ArgCGC8.9010022.2100AGA31.0
ProCCC17.820.01000100CCA40.8
LeuCUG14.15.085.735.8100CUU20.6
GlyGGC16.317.310031.8100GGA34.6
AlaGCC16.122.310040.0100GCU33.5
SerUCC12.711.130.033.4100UCA24.3

We used overlap polymerase chain reaction (PCR; Ho et al.,1989; Horton et al.,1989) to generate three GFP variants, all using the Aequoria GFP sequence as a cloning scaffold: aGFPM5, aBFP, and aCFP. The mutations required to generate each of these variants, and the nomenclature we use to describe the various fluorescent proteins analyzed in this study are described in Table 1. As described in the Experimental Procedures section, the mutations we introduced are similar to previously reported mutations from other laboratories and we are not aware of the commercial availability of any of these variants. The mutations S65T (GFP); Y66H and Y145F (BFP); and Y66W, N146I, and N212K (CFP) are responsible for producing the shifts in excitation and emission spectra, whereas the F64L, M153T, I167T, and S175G mutations are probably responsible for improved protein folding (Tsien,1998). We expressed each protein in Escherichia coli, and the excitation and emission spectra of each of these purified proteins was analyzed in a fluorescent plate reader (Fig. 1). Our GFP variants have spectra nearly identical to previously described BFP, CFP, and GFP variants, and we used these spectra to obtain optimized filter cubes for epifluorescence microscopy (detailed in the Experimental Procedures section). We did not generate any novel variants of DsRed, a fluorescent protein found in an anthozoan (Matz et al.,1999), but we did compare the expression of embryos expressing HDsRed2 (human codon optimized DsRed), wtDsRed (wild-type anthozoan DsRed), and HmRFP (human codon optimized monomeric DsRed).

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Figure 1. Excitation and emission spectra of green fluorescent protein (GFP) variants measured with a scanning fluorescent plate reader. The numbers indicate the peaks of excitation and emission spectra of aBFP (1=EX, 3=EM), aCFP (2=EX, 4=EX) and aGFPM5 (5=EX, 6=EM).

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We generated a series of transgenes expressing our fluorescent protein variants to measure the expression of these proteins in living ascidians during embryonic development. For our comparative analyses, we used the ∼3-kb cis-regulatory domain of the C. intestinalis Brachyury gene (Corbo et al.,1997) to drive fluorescent protein expression in the notochord cells of the embryo. An example of DsRed expression is depicted in Figure 2, where each DsRed variant is expressed in the notochord cells of transgenic embryos. In these transgenic embryos, 50 μg of the reporter gene plasmid was electroporated into dechorionated, fertilized eggs using electroporation parameters of 3,000 μF with a 10 Ohm timing resistor. In qualitative terms, HmRFP fluorescence is detectable in embryos 3–4 hr earlier (at approximately 6 hours postfertilization [hpf]) than either HDsRed2 or wtDsRed (at approximately 10–12 hpf). In addition to the need for proper protein folding and maturation, both wtDsRed and HDsRed2 proteins require tetramerization before they can be detected by epifluorescence microscopy (reviewed by Verkhusha and Lukyanov,2004), likely contributing to the longer time until detection. Because HmRFP, a monomeric form of DsRed (Campbell et al.,2002), was detectable much earlier than the other DsRed variants examined, only the HmRFP was used in the quantitative expression experiments described below.

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Figure 2. Expression of DsRed variants in ascidian embryos. The notochord-specific CiBra transgene was employed to drive expression of DsRed variants in ascidian embryos. A,B: HDsRed2 expression. C,D: wtDsRed expression. E,F: HmRFP expression. A, C, and E are differential interference contrast microscopy images of transgenic embryos. B, D, and F are images of DsRed expression. Expression of HmRFP protein is detected around 5.5 hours postfertilization (hpf), whereas HDsRed2 and wtDsRed are not detected until 10–12 hpf.

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Three of the fluorescent proteins we examined (BFP, GFP, and DsRed) have well separated excitation and emission spectra that can be distinguished with commonly used 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI), fluorescein isothiocyanate (FITC), and tetrarhodamine isothiocyanate (TRITC) filter cubes. The excitation and emission spectra of CFP significantly overlap with the excitation and emission spectra of GFP (Fig. 1); therefore, optimized filter sets were used to reduce this overlap. We electroporated dechorionated fertilized eggs with 50 μg of the CiBra::aCFP transgene (3,000 μF capacitance, 10 Ohm timing resistor) to drive CFP expression in the notochord cells of the ascidian embryo. The differential interference contrast microscopy image of an example 12-hr tadpole larva is shown in Figure 3A, along with images of CFP and GFP expression with standard (Fig. 3B) and optimized (Fig. 3C) CFP filter cubes. All images of fluorescent protein expression were acquired with identical exposure settings. Although the CFP signal is reduced when using the optimized emission filter, which contains a narrow bandpass emission filter, signal contamination with GFP-expressing cells is greatly reduced and nearly eliminated. We do observe some CFP expression when using the GFP filter set, suggesting that our filter sets could be further optimized (data not shown).

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Figure 3. Cyan fluorescent protein (CFP) expression in the notochord of a transgenic ascidian embryo. CFP expression was driven by the 3.5-kb CiBra promoter as described in the Experimental Procedures section. A: Differential interference contrast microscopy image. B: CFP fluorescence with standard filter set. C: CFP image with narrow emission filter. D: CFP fluorescence in green fluorescent protein (GFP) filter cube, indicating a small amount of detectable emission. All fluorescence images were obtained with the exact same exposure settings. There is also some GFP emission bleed through when using the CFP filter set (data not shown).

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To examine the feasibility of imaging multiple fluorescent proteins expressed in a single transgenic embryo, we generated three different tissue-specific reporter genes, each expressing a different fluorescent protein. The cis-regulatory domains for two of these reporter genes, CiBra and HrM, have been described previously and drive reporter expression in the C. intestinalis embryonic notochord and muscle cells, respectively (Corbo et al.,1997). The third reporter gene (BT) was derived from the C. intestinalis orthologue of the Halocynthia roretzi β-tubulin gene as this gene is known to be expressed in the central nervous system of the ascidian larva (Miya and Satoh,1997). The computational methods and cloning strategy for generating this transgene are detailed in the Experimental Procedures section. Equal amounts of the three transgenes (30 μg each of HrM::aBFP, BT::aGFPM5 and CiBra::wtDsRed) were co-electroporated into fertilized ascidian eggs (1,000 μF capacitance, 30 Ohm timing resistor), and transgene expression was observed in 12- to 14-hr-old embryos. Examples of two embryos expressing all three reporter genes are shown in Figure 4. In each embryo, the transgenes are expressed mosaically but in the expected cell types: notochord for CiBra::wtDsRed, muscle for HrM::aBFP, and the central nervous system (CNS) for BT::aGFPM5. In the first embryo (Fig. 4A–D), the HrM::aBFP transgene is expressed in two stripes in the B-lineage primary muscle cells of the tadpole larva. The BT::aGFPM5 transgene is expressed in the larval CNS in the trunk, as well as in the neural tube that extends down the dorsal side of the tail. The CiBra::wtDsRed transgene is expressed in nearly all of the primary notochord cells of the anterior tail as well as in some of the secondary cells in the posterior tail, although the wtDsRed expression in these cells is more difficult to detect. In the second embryo (Fig. 4E–H), all three transgenes, CiBra::wtDsRed, HrM::aBFP, and BT::aGFPM5, are expressed in approximately 50% of the expected cells. These fluorescent proteins are suitable for the simultaneous imaging of three different transgenes in living ascidian embryos; however, it should be noted that aBFP photobleaches significantly faster than either aCFP or aGFPM5.

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Figure 4. A–H: Transgenic ascidian embryos simultaneously expressing different fluorescent proteins under the control of three different transgenes. Equal amounts of the three transgenes (30 μg) were introduced into fertilized eggs by electroporation. A–D,E–H: Two example embryos are shown. A,E: Differential interference contrast microscopy images. B,F: HrM::aBFP expression. C,G: BT::aGFPM5 expression. D,H: CiBra::wtDsRed expression. All transgenes are expressed in the expected cell types: central nervous system for BT, muscle for HrM, and notochord for CiBra.

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Fluorescent Proteins, Codon Optimized for Translation in Ciona, Are Efficiently Expressed in Transgenic Embryos

Previous studies have demonstrated that the expression of fluorescent proteins (Yang et al.,1996), as well as many other kinds of proteins (Gustafsson et al.,2004) is significantly enhanced by optimizing the codons for specific cell types, such as human cells. Over the past few years, our own experiences in expressing fluorescent proteins in transgenic ascidian embryos have supported this hypothesis. To determine whether fluorescent protein expression and detection in transgenic ascidian embryos may be further improved, we generated codon optimized variants of CFP (CoCFP), GFP (CoGFP), and mRFP (ComRFP) as described in the Experimental Procedures section. For both GFP (aGFPM5 and CoGFPM5) and mRFP (HmRFP and ComRFP) variants, the amino acid sequences are identical (Table 1), thus any alterations in the expression and detection of these proteins are due to the altered codons in the mRNAs encoding the fluorescent proteins. For CFP, additional mutations were incorporated in the optimized variant (Table 1), thus changes in expression are likely due to codon optimization as well as the introduced amino acid changes. It should be noted that our purpose in this experiment was not to rigorously test fluorescent proteins of identical amino sequence for effects of codon optimization, but simply to generate the best possible fluorescent proteins for expression in transgenic ascidians.

To quantitatively test the expression of the optimized CFP, GFP, and mRFP variants, we generated transgenes in which the coding region for each fluorescent protein was attached to the same ∼3-kb CiBra cis-regulatory region previously described. For each transgene, 50 μg of plasmid DNA was electroporated into dechorionated fertilized eggs (settings 1,000 μF, 30 Ohm timing resistor) and time-lapse movies were recorded from approximately 200 to 800 min after fertilization. Images were acquired every 5 min during the time-lapse, and the same exposure time (200 msec) was used in each time-lapse. Two movies were generated for each construct, and mean and maximum pixel intensities for individual embryos were measured over the entire time-lapse period as described in the Experimental Procedures section. A summary of these measurements is shown in Figure 5, where the normalized average maximum and mean pixel values per embryo are plotted vs. time after fertilization. To determine whether there were differences between the fluorescent protein variants, a plot of pixel value vs. time, for each individual embryo, was generated and the area under this curve was calculated. This calculation provides a measurement of fluorescent protein expression over the time course of the experiment on a per embryo basis. An analysis of variance (ANOVA) analysis was then used to determine whether there were significant differences between the expression of the different fluorescent proteins (Table 3). Because pixel threshold values were used to help automate the analysis of the time-lapse images, it was difficult to measure accurately the very early expression of the fluorescent proteins. By carefully analyzing the first few early time-lapse frames individually, we could first detect fluorescence from the various proteins as follows: HmRFP and ComRFP (∼350 min), wtCFP (∼385 min), CoCFP (∼280 min), HeGFP (∼280 min), aGFPM5 (∼280 min), and CoGFPM5 (∼260) min. For reference, the Brachyury gene is first expressed in C. intestinalis around the 44- to 64-cell stage, approximately 220 min after fertilization.

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Figure 5. Quantitative analysis of time-lapse movies of fluorescent protein expression in transgenic Ciona intestinalis embryos. Each panel depicts the average pixel intensity per embryo vs. time post fertilization in minutes. The pixel values are normalized and the largest measurement is defined as 100%. Panels labeled “mean” report average mean pixel values per embryo, and panels labeled “max” report average maximum pixel values per embryo. Squares, Ciona codon optimized proteins (CoCFP, CoGFPM5 and ComRFP); circles, aCFP and aGFPM5; and triangles, HmRFP and HeGFP.

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Table 3. ANOVA Analysis Summary of Transgene Time-Lapse Recordingsa
Transgene comparisonP value
  • a

    Mean or maximum pixel intensity is per embryo. For GFP measurements, low threshold refers to measurements made prior to ∼500 min postfertilization when background pixel values are low. High threshold measurements do not accurately measure fluorescence prior to 500 min postfertilization. The numbers in parentheses indicate the sample size of embryos analyzed for a particular construct. N.S., not significant. ANOVA, analysis of variance; GFP, green fluorescent protein.

ComRFP (n = 31) vs. HmRFP (n = 31) mean or maximum pixel intensityN.S.
CoCFP (n = 33) vs. aCFP (n = 72) mean or maximum pixel intensity< 0.01
Low threshold measurement maximum pixel intensity 
CoGFPM5 (n = 33) vs. aGFPM5 (n = 36)N.S.
CoGFPM5 (n = 33) vs. HeGFP (n = 34)< 0.01
aGFPM5 (n = 36) vs. HeGFP (n = 34)N.S.
Low threshold measurement mean pixel intensity 
CoGFPM5 (n = 33) vs. aGFPM5 (n = 36)< 0.01
CoGFPM5 (n = 33) vs. HeGFP (n = 34)< 0.02
aGFPM5 (n = 36) vs. HeGFP (n = 34)N.S.
High threshold measurement maximum pixel intensity 
CoGFPM5 (n = 33) vs. aGFPM5 (n = 36)< 0.01
CoGFPM5 (n = 33) vs. HeGFP (n = 34)< 0.01
aGFPM5 (n = 36) vs. HeGFP (n = 34)< 0.01
High threshold measurement mean pixel intensity 
CoGFPM5 (n = 33) vs. aGFPM5 (36)< 0.01
CoGFPM5 (n = 33) vs. HeGFP (n = 34)< 0.01
aGFPM5 (n = 36) vs. HeGFP (n = 34)< 0.01

From the data presented in Figure 5 and Table 3, several conclusions may be made. The codon optimization and altered amino acids present in the CoCFP variant result in the earlier detection (by approximately 90 min) and higher level of expression (judged by pixel intensity) of CoCFP compared with aCFP (P < 0.01). By approximately 12 hpf, average mean pixel values per embryo are over 50% greater for CoCFP compared with aCFP and average maximum pixel values are approximately fivefold greater for CoCFP compared with aCFP. We found no significant difference between the expression of HmRFP and ComRFP (Table 3); however, embryos expressing ComRFP have higher average pixel values at the end of the time-lapse then embryos expressed HmRFP (Fig. 5).

Two separate measurements were required to measure GFP expression in which the lower threshold pixel value was set to either a low (300) or high (450) value because background intensity increased during the course of the time-lapse as a result of reflection from the bottom of the culture dish. The low threshold measurements are more accurate for the time course from 200–500 min after fertilization, whereas the high threshold measurements are more accurate from approximately 500–800 min, when the background pixel intensity was higher. From the low threshold analysis, maximum pixel intensity, only the expression of codon optimized GFP (CoGFPM5) is significantly different than HeGFP expression (P < 0.01). When comparing mean pixel intensity, low threshold analysis, CoGFPM5 expression is significantly different from aGFPM5 expression (P < 0.01) and from HeGFP expression (P < 0.02). Expression of all GFP variants was significantly different from one another when the data were analyzed using the high pixel threshold value (P < 0.01).

Subcellular Localization of Fluorescent Proteins to the Nucleus and Golgi

Subcellular structures may be observed in living cells and embryos by fusing the coding regions of fluorescent proteins to appropriate localization sequences. We, therefore, tested the ability of our fluorescent proteins to localize to two different subcellular compartments—the nucleus and the Golgi apparatus. We combined the aGFPM5 coding sequence, the lacZ coding sequence, and the SV40 NLS to produce a fusion protein that localized primarily to the nucleus (Fig. 6A,B). Expression of transgenes using this reporter gene can be detected visually in living embryos (GFP fluorescence) or can be detected by the histochemical detection of lacZ for more sensitive detection. We also generated a nuclear-localized reporter by creating a translational fusion between the C. intestinalis histone H2A gene and the HmRFP gene and expressed this reporter gene under the control of the C. intestinalis EpiB cis-regulatory domain. When expressed in transgenic embryos, this reporter gene provides a robust nuclear localization that is superior to the NLS-GFP-lacZ fusion protein as shown in an optical section (Fig. 6C). In general, the histochemical staining of lacZ is more sensitive than GFP expression due to the enzymatic amplification of the reaction products, which can be allowed to accumulate over time. The use of this dual reporter molecule simplifies transgene construction because a single construct may express a fluorescent protein with lacZ enzymatic activity.

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Figure 6. Nuclear localization of fluorescent proteins in transgenic ascidian embryos. A,B: Fertilized eggs were electroporated with the CiBra promoter driving expression of the aGFPM5-lacZ reporter gene. A: Green fluorescent protein (GFP) expression. B: Staining for lacZ activity in the same batch of embryos shown in A. In both examples, the SV40 NLS localizes most of the fusion protein to the cell nucleus, but there still remains significant cytoplasmic protein. C: Optical section of a transgenic embryo expressing the CiEpiB::H2A-HmRFP transgene. When expressed, the histone H2A-HmRFP fusion protein is present in the cell nucleus providing improved nuclear localization compared to the aGFPM5-lacZ fusion protein.

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By fusing a short peptide tag from the sialyltransferase gene to GFP, GFP may be localized to the Golgi apparatus as previously demonstrated in plants (Boevink et al.,1998). We created a Golgi-localized fluorescent protein by attaching the amino terminal anchoring sequence from sialyltransferase to aGFPM5 and expressed this fusion protein in the notochord cells of the ascidian embryo under the transcriptional control of the CiBra promoter (Fig. 7). One of the hallmark morphological changes during notochord differentiation in C. intestinalis is the production of vacuoles that will eventually form the lumen of the notochord (Miyamoto and Crowther,1985; Cloney,1990), and electron photomicrographs have demonstrated that the Golgi is closely associated with these vacuoles and is responsible for packaging matrix colloid material and membrane leaflets (Crowther and Whittaker,1986). When the CiBra::ST-GFPM5 DNA is electroporated into fertilized eggs, the resulting embryos express the transgene, fluorescence is detected in a subcellular pattern that is consistent with the localization of the Golgi. Several different embryos expressing this transgene over a time course experiment are shown in Figure 7 and include embryos at 11.5 hr (Fig. 7A,D), 12.5 hr (Fig. 7B,E), 14 hr (Fig. 7C,F), and 16 hr (Fig. 7G–I). The notochord cells assume a “stack of coins” configuration by approximately 11 hr (Miyamoto and Crowther,1985; Cloney,1990), and the Golgi in these cells is localized to opposite sides of the cells and appears in a striped pattern. As development proceeds and the vacuoles begin to increase in size, the Golgi changes position within the cell, eventually becoming closely juxtaposed to the vacuoles as can be seen in Figure 7G–I.

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Figure 7. Subcellular localization of ST-aGFPM5 to the Golgi apparatus is consistent with previous observations that the Golgi is important for notochord differentiation. Fertilized eggs were electroporated with the transgene CiBra::ST-aGFPM5, a green fluorescent protein (GFP) fusion protein that localizes to the Golgi. A–F: Example embryos from three different time points (11.5 hr, A,D; 12 hr, B,E; and 14 hr, C,F). AC: differential interference contrast microscopy images. DF: Corresponding GFP images. GI: The 16 hr embryos expressing the same transgene. As development proceeds, the notochord cells begin to produce vacuoles and secrete extracellular matrix materials. As the vacuoles begin to form, the Golgi translocates from a position on opposite sides of each cell (D–E) to a position surrounding the vacuoles (F–I). In all images, black arrows indicate the notochord vacuoles and white arrowheads indicate the Golgi. Notochord cell nuclei are not visible.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In the present study, we demonstrated that, while commercially available fluorescent proteins can be expressed and detected in ascidian embryos, the genes encoding these proteins are not optimized for maximum expression and detection. In our experiences expressing both GFP and DsRed in transgenic ascidian embryos, we have consistently observed that the humanized versions of these proteins are not expressed as well as the variants of these proteins generated with an Aequoria sequence template. Our observations are corroborated by the codon usage analysis we undertook by comparing the frequency of codons used in ascidian genes with the optimized codons selected for the “humanized” fluorescent protein variants. To maximize the expression of fluorescent proteins in ascidians, we generated codon optimized genes encoding CFP, GFP, and mRFP. Our quantitative analysis of expression data indicates that expression of our optimized CFP clone was detectable by epifluorescence microscopy approximately 90 min before expression of our original CFP clone. Expression of our optimized GFP (CoGFPM5) was detectable within 20 min of HeGFP and aGFPM5, and at the end of the time lapse, the mean pixel value of embryos expressing CoGFPM5 was approximately 50% greater then embryos expressing HeGFP. Although these proteins are all detected within 20 min of each other, the slope of the pixel intensity plot of embryos expressing CoGFPM5 is greater than either aGFPM5 or HeGFP, suggesting that CoGFPM5 fluorescence is appearing at a more rapid rate. We measured no significant difference in the expression of our mRFP variants, although they both expressed at much higher levels and were detected much earlier than either wtDsRed or HDsRed2. By the end of the time lapse recordings, maximal pixel intensities for HmRFP expression were only 80% of ComRFP expression. With the exception of the mRFP variant, our codon optimized CFP and GFP variants (CoCFP and CoGFPM5) are superior to the clones our laboratory has been using for several years now (aCFP and aGFPM5).

The use of humanized versions of fluorescent proteins may pose no major problems for routine use in transgenic ascidian embryos, particularly when fluorescent protein expression is assayed at the end of embryonic development. However, in some experimental situations, it may be desirable to observe GFP expression as early as possible or at maximal detectable levels, such as when recording a movie of GFP expression during development, and it will be necessary to use all practical means to optimize GFP expression and detection. Previous studies have demonstrated that GFP detection in living cells is possible only after sufficient numbers of GFP molecules are synthesized. In the zebrafish embryo, approximately 105 molecules per cell are required for detection in singly labeled embryonic cells (Amsterdam et al.,1996). In many cell types, there is significant autofluorescence that requires micromolar amounts of GFP to be present to observe GFP fluorescence (Niswender et al.,1995). One of the ways in which protein expression may be maximized is by creating coding variants that only use the most frequently used codons in the particular cell type expressing GFP, as was reported for the humanized version of GFP (Yang et al.,1996). Additionally, research from other laboratories continues to identify new amino acid alterations that increase the efficiency of fluorescent protein expression and detection (Tsien,1998). Importantly, we demonstrated that a combination of codon optimization and amino acid substitutions generated several fluorescent protein genes optimized for expression in Ciona embryos.

We created transgenic ascidian embryos expressing three different fluorescent protein transgenes and showed that we could detect each of these proteins in the same embryo. We were unable to completely eliminate interference between GFP and CFP emissions, but others have demonstrated that it possible to do so, at least in bacterial cells (Heim and Tsien,1996), and a modification of our filter sets may further improve our ability to distinguish the expression of these two fluorescent proteins. Because aBFP photobleaches rapidly compared with our other proteins, we have started to use the Venus variant of YFP (Nagai et al.,2002) in combination with CoCFP and ComRFP. Emission spectra from embryos expressing these three fluorescent proteins are more widely separated for easier detection, and there is no longer an issue with aBFP photobleaching. It should be noted that a standard epifluorescence microscope, equipped with the proper filters sets, is all that is required to image our three fluorescent proteins expressed together in single embryos. Lastly, we show that our fluorescent proteins, with the appropriate localization tags, may be efficiently localized to subcellular compartments and that a dual-reporter GFP-lacZ fusion protein may simplify transgene construction.

The ability to simultaneously express and detect multiple fluorescent proteins in living embryos raises the possibility of recording quantitative measurements of the expression of three or four genes during embryonic development. Recent techniques have been developed to accurately quantitate GFP expression in living sea urchin embryos as well as in cell lysates from these embryos (Dmochowski et al.,2002) or other types of cells (Albano et al.,1998). Despite the initial success of these experiments, fluorescent proteins present a problem—they are extremely resistant to degradation and, therefore, are quite stable within cells (Chalfie et al.,1994). To more accurately measure transcriptional activity, the half-lives of the fluorescent proteins must be reduced, and at the same time, optimizations to maximize the expression of properly folded protein must be maintained. Recent experiments have created “destabilized” GFP variants with reduced half-lives by fusing protein domains that will target GFP for degradation (Li et al.,1998; Corish and Tyler-Smith,1999). By creating destabilized, codon-optimized fluorescent proteins with different spectral properties, the quantitative measurements of multiple genes expressed simultaneously in living embryos may soon become a reality.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Embryo Culturing and Electroporation Procedure

Adult Ciona intestinalis were collected in various marinas located in San Diego County, California, or in Woods Hole, Massachusetts. All manipulations were performed at 18°C in water baths or on copper cold plates. Under these conditions, control embryos hatch in 18 hr postfertilization (hpf), and in our experience, maintaining controlled temperatures greatly enhances the ability to generate transgenic embryos.

A detailed electroporation procedure, based on the original protocol (Corbo et al.,1997) is described elsewhere (Zeller,2004). Two different custom-built electroporators were used to introduce exogenous DNA into fertilized dechorionated eggs (Zeller et al., submitted). Using these devices, the electroporation parameter of 1,000 μF capacitance with a 30 Ohm timing resistor most closely approximates the settings reported by (Corbo et al.,1997). Some of the electroporations (indicated in text) were performed with a setting of 3,000 μF with a 10 Ohm timing resistor; a parameter that generally allows for greater than 50% normal embryonic development (compared with control dechorionated, but not electroporated, embryos) with minimal mosaic transgene expression. All embryos reported in this study are most likely expressing GFP transgenes transiently (we did not test if the transgenes integration into the genome), although electroporation may be used to generate stable transgenic ascidians (Matsuoka et al.,2005). Unless otherwise stated, DNA concentration was 50 μg per 800-μl electroporation. In the set of experiments in which three transgenes were co-electroporated into fertilized eggs, 30 μg of each transgene was used in an 800-μl electroporation. Transgenic embryos were reared in 0.45 μM filtered sea water containing 0.1 mM ethylenediaminetetraacetic acid and antibiotics (10 U of penicillin and 10 μg of streptomycin per ml of sea water).

Site-Directed Mutagenesis and Codon Optimization

Overlap PCR (Ho et al.,1989; Horton et al.,1989) was used to introduce all of the point mutations into the GFP coding sequence. PCR templates were wild-type GFP, clone P4-3, or clone W7, kindly provided by Dr. Roger Tsien, UC San Diego. To our knowledge, the fluorescent protein variants we generated, in a wild-type GFP backbone, are not commercially available. The GFP and BFP sequences are similar those previously reported (Zernicka-Goetz et al.,1996), and the final CFP we used is similar to a variant called cerulean (Rizzo et al.,2004). An internal EcoRI restriction site present in wild-type DsRed was removed by overlap PCR without altering any codons. Table 1 and Supplementary Figure S1 (which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat) describe the mutations introduced into the GFP variants. Clones for three additional proteins are distributed by Clontech (Palo Alto, CA): humanized versions of enhanced GFP and DsRed (HeGFP and HDsRed2, respectively), and wild-type (wtDsRed). A monomeric form of DsRed (HmRFP; Campbell et al.,2002) was kindly provided by Dr. Roger Tsien.

Codon optimization was carried out on a fee for service basis by DNA 2.0 (http://www.dnatwopointo.com, Menlo Park, CA) using the Ciona intestinalis codon usage table from the codon usage database (http://www.kazusa.or.jp/codon/). The Ciona optimized sequences contain not only codon alterations to eliminate the use of rare codons, but also changes in codons designed to eliminate unwanted mRNA secondary structure and restriction enzyme sites (Gustafsson et al.,2004). Table 1 describes the notation for the various fluorescent proteins used in this study. GenBank accession numbers for codon-optimized proteins are as follows: optimized GFP (DQ229367); optimized CFP (DQ229368) and optimized mRFP (DQ229369).

Protein Expression, Purification, and Determination of Excitation and Emission Spectra

DNAs encoding all GFP variants were subcloned into the prokaryotic expression vector pTrcHisC (Invitrogen, Carlsbad, CA), induced with 1mM IPTG and purified on NTA-nickel resin according to the manufacturer's instructions (Qiagen, Valencia, CA). Excitation and emission spectra (Fig. 2) were determined on dilution series of each GFP variant by using a SpectraMax Gemini XS fluorescent plate reader according to the manufacturer's instructions (Molecular Devices, Sunnyvale, CA).

Transgene Construction

We have standardized our transgene construction using the vector pSP72 (Promega), and our electroporation vector is shown in Supplementary Figure S2. In general, the cis-regulatory domains are PCR amplified as XhoI–Asp718 fragments, and the reporter gene coding sequence is amplified as an Asp718-EcoRI fragment. We generally make translational fusions that incorporate the first 8–10 amino acids of the endogenous gene. Some of the transgenes used in this study have been described previously—the C. intestinalis Brachyury transgene (CiBra) and the Halocynthia roretzi muscle actin transgene (HrM) by Corbo et al.,1997). The cis-regulatory domains from these genes were used to create CiBra::aGFPM5, CiBra::CoGFPM5, CiBra::HeGFP, CiBra::aCFP, CiBra::CoCFP, CiBra::HDsRed2, CiBra::wtDsRed, CiBra::HmRFP, CiBra::ComRFP and HrM::aBFP.

Oligonucleotides used to generate additional transgenes included (genomic DNA sequences are capitalized; lower case sequences indicate restriction enzyme sites incorporated into the PCR primers): BT 5p, gcgctcgagAGACGACGCCGTAAAGTTGT; BT 3p, gcgtggtaccGCTTGCAAATGAACAATTTCTCTCAT; CiEpiB 5p, gcgctcgagTTTATACATTGCAGTCAAACGAAGCACC; CiEpiB 3p, cgcggtaccATTTTACTATTTTAATTACG; H2A 5p, cgcggtaccaATGGCTGGAGGCAAAGCAGG; H2A 3p, cgcgttaccTTTGTATAGTTCATCCATGCC.

The β-tubulin transgene BT::aGFPM5 contains a ∼3-kb genomic fragment PCR amplified from C. intestinalis genomic DNA that fuses the second of two alternative 5′ exons to GFP (Supplementary Figure S3). The CiEpiB transgene contains a ∼2-kb genomic fragment, and the reporter gene in this construct (CiEpiB::H2A-HmRFP) consists of an Asp718–BamHI fragment of the C. intestinalis histone H2A variant Z gene (JGI predicted gene ci0100134847) fused to a BamHI–EcoRI fragment of HmRFP. To make the aGFPM5-lacZ fusion reporter gene, PCR amplification was used to introduce NheI sites in the aGFPM5 and lacZ coding sequences. The SV40 nuclear localization signal (NLS) from pSP72-1.27 (Fire et al.,1990) was inserted at the 5′ ASP718 site to create NLS-aGFPM5-lacZ. The membrane signal anchor sequence from rat sialyl transferase (Boevink et al.,1998) was fused to the amino terminal end of aGFPM5, by means of PCR, to create ST-aGFPM5 that localizes to the Golgi.

Computational Methods

We conducted a BLAST search (Altschul et al.,1990) to identify C. intestinalis orthologues of the Halocynthia roretzi β-tubulin gene (Miya and Satoh,1997). Expression patterns archived at the Kyoto EST resource site (http://ghost.zool.kyoto-u.ac.jp/indexr1.html) confirmed that this gene (EST cluster 00086, JGI predicted gene ci0100135737) was expressed in the central nervous system (CNS). The region of genomic sequence containing the entire β-tubulin gene up to the flanking 5′ and 3′ predicted genes for both C. intestinalis and C. savignyi genome were analyzed with the mVISTA webserver (http://www-gsd.lbl.gov/vista/; Mayor et al.,2000; Loots et al.,2002) for phylogenetic footprint analysis (Supplementary Figure S3). PCR primers, listed above, were used to amplify a ∼3-kb region of the of β-tubulin gene. The C. intestinalis EpiB cis-regulatory domain (CiEpiB, JGI predicted gene ci0100132755) was identified and cloned in a similar manner.

Microscopy and Image Analysis/Processing

Images were obtained using the following equipment: a Nikon Coolpix 995 camera or a Zeiss AxioCam HRm camera mounted on a Zeiss Axioplan 2e Imaging microscope equipped with an ApoTome for collecting optical sections. Microscope control was provided by AxioVision v4.2. Adobe Photoshop was used to create composite and overlay images. Optimized fluorescent microscopy filter sets were obtained from Chroma Technology Corp. (Rockingham, VT). Optimized filter sets were BFP set #31041, CFP set #31044v2, GFP set #41017, and CFP narrow band emission filter 470/20.

Seven different reporter transgenes expressing a different fluorescent protein were analyzed in duplicate by making time-lapse recordings, one image acquired every 5 min, of embryos expressing fluorescent proteins from approximately 200–800 min postfertilization. For each construct, 50 μg of plasmid DNA was electroporated into fertilized, dechorionated eggs (settings 1,000 μF, 30 Ohm resister). Embryos were imaged as they developed inside a gelatin-coated 60-mm culture dish. Images were acquired with a Zeiss AxioCam HRm 14-bit digital camera using a fixed exposure time of 200 msec. Using this camera, pixels had a range of values from 0 to 16,383. Background pixel values ranged from 250–450, and some pixels became saturated (values > 16,383) by the end of the recordings. Each time-lapse recording was imported into ImageJ (http://rsb.info.nih.gov/ij/) as a stack and threshold pixel values were established that accurately detected only fluorescence from the expression of the transgenes and not from background. Mean and maximum pixel values, representing fluorescent protein expression, were measured for individual embryos within the time-lapse recordings. For each embryo, a plot of pixel value vs. time was generated and the area under this curve was used as a measurement of fluorescent protein expression over the course of the experiment on a per embryo basis. An ANOVA analysis was used to analyze statistically significant differences among these embryo populations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Dr. Roger Tsien and Dr. Nori Satoh for providing plasmids and clones. We thank Dr. Anca Segall for the use of the fluorescent plate reader. R.W.Z. was funded by a grant from the San Diego State University Center for Applied and Experimental Genomics, a CSUPERB Faculty Seed Grant, and NSF Career Grant.

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  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/1058-8388/suppmat

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jws-dvdy.20644.fig3.tif305KSupporting Information file jws-dvdy.20644.fig3.tif

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